Herbal based composition for treating acute and chronic myeloid leukemia

ABSTRACT

A new herbal-based composition and method for treatment of CD33+ acute and chronic myeloid leukemia by  Piper betel  leaf extracts, and to provide a process for the isolation of active fractions from leaves or any other plant parts of  Piper betel  to treat CD3 3+ AML and CML with a simplified method of isolation of active components from all plant parts of  Piper betel  possessing biological activities relevant to the treatment of CD33+ AML and CML.

[0001] This application claims benefit of U.S. Provisional ApplicationNo. 60/384,163 filed May 31, 2002

FIELD OF INVENTION

[0002] This invention relates to a herbal based composition fortreatment of CD33+ acute and chronic myeloid leukemia by Piper betelleaf extracts, fractions of Piper betel leaf extracts and 3-O-p-coumarylquinic acid purified from Piper betel leaves extract.

BACKGROUND AND PRIOR ART REFERENCES

[0003] Mycloid leukemia, both acute (AML) and chronic (CML) are lethal,there is no drug directing towards the destruction of the myeloid cellsand these cells poorly respond to chemotherapy, which is alwaysnon-specific, thus adversely affecting normal cells. Unique property ofthe therapy with Piper betel components is the killing of myeloid cancercells by recognizing CD33+ marker on the cell membrane, leaving normalcells without getting affected.

[0004] Myeloid leukemia is usually subdivided into two groups: AcuteMyeloid Leukemia (AML) and Chronic Myeloid Leukemia (CML). AML ischaracterized by an increase in the number of myeloid cells in the bonemarrow and an arrest in their maturation. In the United States, theannual incidence of AML is approximately 2.4 per 100,000 and itincreases progressively with age, to a peak of 12.6 per 100,000 inadults of 65 years age or older. The CML is a malignant clonal disorderof hematopoietic stem cells. The median age at presentation is 53 years,but it occurs at all age groups, including children. The natural historyof CML is progression from a benign chronic phase to a rapidly fatalblast crisis within three to five years or even earlier. The prognosisof CML is also poor inspite of vast advancement of clinical medicine(1). CD33 represents a specific and useful marker in the process ofmyeloid cell differentiation (2). Recent reports suggest that engagementof CD33 by monoclonal antibody induced apoptosis leading to growthinhibition of proliferation of AML and CML cells in vitro (2,3).Exploiting the myeloid specific expression of CD33, humanized anti-CD33monoclonal antibody conjugated with anti-cancer drug has been tried inAML patients with significant success (4). With the extract from Piperbetel leaves anti-myeloid activity was claimed earlier (Patent filed no.PCT/INOO/00118 dated Dec. 12, 2000).

[0005] Hence, applicant's earlier finding is in directed consonance withthe present patent filing on Piper betel leaf extract, fractionated leafextracts and purified compound 3-O-p-coumaryl quinic acid (FIG. 1)obtained from leaf extract for treating CD33+ acute and chronic myeloidleukemia.

[0006]Piper betel leaves have a strong pungent aromatic flavour and arewidely used in India as a masticatory. Generally, mature or overmatureleaves, which have ceased growing but not yet become brittle are usedfor chewing. The basic preparation for chewing purposes consists ofbetel leaf smeared with hydrated lime and catechu to which scrapings ofarecanut are added; flavourings such as coconut shavings, clove,cardamom, fennel, powdered liquorice, nutmeg and also tobacco are usedaccording to one's taste. In some places prepared Piper betel leafpreparation is covered with silver or gold film. As a masticatory, it iscredited with many properties: it is aromatic, digestive, stimulant andcarminative. Medicinally it is useful in catarly and pulmonaryinfections; it is also uses for poultices. The effects of chewing ofbetel leaves with arecanut and other adjuncts are the excitation of thesalivary glands and the irritation of the mucous membrane of the mouth.The red coloration produced is due to a pigment in the arecanut, whichmanifests itself under the action of alkali in lime and catchu. A milddegree of stimulation is produced, resulting in a sensation of warmthand well-being, besides imparting a pleasant odour. The most importantfactor determining the aromatic value of the leaf is the amount andparticularly the nature of the essential oil present. Betel leaves fromdifferent regions vary in smell and taste. The most pungent is theSanchi type, while the most wild and sweet ones are from Varansai. Thebetal leaves contain essential oils, the content of oil varies from 0.7to 2.6 per cent depending upon the varieties of leaves. The oil consistsof phenols and terpens. The higher the proportion of phenol the betterthe quality. An isomer of eugenol named chavibetol (betel phenol;4-alkyl-2-hydroxy-1methoxy benzene) is considered to be characteristicconstituent of betel oil. Betel oil of Indian types contains apredominant phenolic constitutent and used in the treatment of variousrespiratory problems, either as a local application or by gargle. It hascarminative properties. It exhibits in different actions on the centralnervous system of mammals. The essential oil and extracts of the leavespossess activity against several Gram-positive and Gram-negativebacteria such as Micrococcus pyogenes var. Albus, Bacillus subtilis andB. Megaterium, Diplococcus pneumoniae, Streptococcus pyogenes,Escherichia coli, Salmonella typhosa, Vibrio comma, Shigelladysenteriae, Proteus vulgaris, Pseudomonas solanacaerum, Sarcina luteaand Erwinia carototora. The essential oil and leaf extracts also showedanti-fungal activity against Asperigillus niger and A. Oryzae,Curvularia lunata and Fusarium oxysporum. The oil is found to be lethalin about 5 miutes to the protozoa, Paramaeceum caudatum (5).Steam-distillate of the leaves showed activity against Mycobacteriumtyberculosis.

REFERENCES

[0007] 1. Sawyers C L, The New England Journal of Medicine, 340 (17),1330-1999.

[0008] 2. Vitale, C; Romagnani, C, et al., Proc. Natl. Acd. Sci. USA, 96(26), 15091-15096, 1999.

[0009] 3. Vitale, C et al., Proc. Natl. Acd. Sci, USA, 98 (10),5764-5769, 2001.

[0010] 4. Sievers E L, Appelbaum, F R et al., Blood, 93, 3678-3684,1999.

OBJECT OF THE INVENTION

[0011] The main object of the invention is to provide a new herbal-basedcomposition for the treatment of CD33+ acute and chronic myeloidleukemia by Piper betel leaf extracts.

[0012] Another object of the invention is to provide a process for theisolation of active fractions from leaves or any of the plant parts ofPiper betel to treat CD33+ AML and CML.

[0013] Still another object of the invention is to provide a simplifiedmethod of isolation of active components from all plant parts of Piperbetel possessing biological activities relevant to the treatment ofCD33+ AML and CML.

[0014] Yet another object of the invention is to provide a new herbalproduct from active fraction from leaves or any other plant parts ofPiper betel for the treatment of CD33+ AML and CML.

[0015] Yet another object of the invention is to provide a herbalcompound 3-O-p-coumaryl quinic acid purified from leaves of Piper betelfor the treatment of CD33+ AML and CML.

[0016] Yet another object of the invention is to provide a process forthe preparation of extract from leaves or any other plant parts of Piperbetel for the treatment of CD33+ AML and CML.

[0017] Yet another object of the invention is to provide a simplifiedmethod of extract preparation from leaves or any other plant parts ofPiper betel for the treatment of CD33+ AML and CML.

[0018] Yet another object of the invention is to provide a process forthe preparation of 3-O-p-coumaryl quinic acid from leaves of Piper betelfor the treatment of CD33+ AML and CML.

[0019] Yet another object of the invention is to provide a simplifiedmethod of preparation of 3-O-p-coumaryl quinic acid from all plant partsof Piper betel for the treatment of CD33+ AML and CML.

DESCRIPTION OF THE INVENTION

[0020] Accordingly, the present invention provides an herbal-basedcomposition from piper betle leaves for the treatment of CD33+ acute andchronic myeloid leukemia.

[0021] Doses for myeloid leukemia patient depends on the in-vitroefficiency of the drug from piper betel either individually or incombination, the doses of in-vitro application for human myeloid cancerpatients may range from 200 mg to 500 mg per day per patient if given asindividual fraction (fraction 1 or fraction 9) or as a combination offraction 1 and fraction 9 in the ratio between 0.1:5 to 5.0.1 preferablyin the ratio 1:1 totaling 125 mg to 370 mg per day per patient throughintravenous route.

[0022] In an embodiment of the invention relates to evaluation ofbioactivity of fractions 1 to 9 isolated from piper betel leaf againstcancer cells.

[0023] In an embodiment of the invention relates to evaluation ofbioactivity of fractions 1 to 9 isolated from piper betel leaf for thetreatment of CD33+ acute and chronic myeloid leukemia.

[0024] The invention also provides a process for isolation of3-O-p-coumaryl quinic acid from piper betel, which is reported for thefirst time.

[0025] Another embodiment of the invention relates to a process for theisolation of active fractions from leaves or any other plant parts ofPiper betel to treat CD33+ AML and CML.

[0026] Still another embodiment of the invention relates to a simplifiedmethod of isolation of active components from all plant parts of Piperbetel possessing biological activities relevant to the treatment ofCD33+ AML and CML.

[0027] Yet another embodiment of the invention relates to a new herbalcomposition/fraction from leaves or any other plant parts of Piper betelfor the treatment of CD33+ AML and CML.

[0028] Yet another embodiment of the invention relates to new activityof compound 3-O-p-coumaryl quinic acid purified from leaves of Piperbetel for the treatment of CD33+ AML and CML.

[0029] Yet another embodiment of the invention relates to a process forthe preparation of extract from leaves or any other plant parts of Piperbetel for the treatment of CD33+ AML and CML.

[0030] Yet another embodiment of the invention relates to simplifiedmethod of extract preparation from leaves or any other plant parts ofPiper betel for the treatment of CD33+ AML and CML.

[0031] Yet another embodiment of the invention relates to a process forthe preparation of 3-O-p-coumaryl quinic acid from leaves of Piper betelfor the treatment of CD33+ AML and CML.

[0032] Yet another embodiment of the invention relates to a simplifiedmethod of preparation of 3-O-p-coumaryl quinic acid from all plant partsof Piper betel for the treatment of CD33+ AML and CML.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

[0033]FIG. 1 represents HPLC analysis of fraction E. The first peakhaving retention time 3.6 min is designated as fraction 1, retentiontime 24.0 is designated as fraction 9.

[0034]FIG. 2 represents structure of fraction 9 deduced as3-O-p-coumaryl quinic acid.

[0035]FIG. 3 represents destruction of CD33+ myeloid leukemia cells invitro by P. Betel leaf extract as demonstrated by flow cytometry. Datais presented as histograms. In upper panels, Forward Scattering of light(X-axis) is plotted against the cell numbers (counts Y-axis). In thelower panels, side scattering of light (X-axis) is plotted against thecell numbers. Solid lines represent data with myeloid leukemia cellsbefore treatment with P. betel leaf extract and the dotted linesrepresent data with myeloid leukemiccells after treatment with P. betelleaf extract (10.0 mg/ml) for48 hours.

[0036]FIG. 4 represents destruction of CD33⁺ myeloid leukemic cells invitro by fraction E as demonstrated by flow cytometry. Dot plots ofForward Scatter vs. Side Scatter with CD33⁺ myeloid leukemic cellsbefore and after treatment with fraction E (100.0 μg/ml) for 48 hoursare shown in left panels where gated region (R1) represents myeloidleukemic cells. Two colour dot plots are also shown after staining withfluorescein isothiocyanate (FITC) conjugated anti-CD33 and phycocrytbrin(PE) conjugated anti-CD13 monoclonal antibodies. Isotype matched controlantibodies (FITC and PE conjugated) were used as negative controls.Upper left quadrants represented CD13, CD33⁺ cells and upper rightquadrants represented CD13⁺, CD33⁺ cells.

[0037]FIG. 5 represents CD33⁻ myeloid leukemiccells are unaffected byfraction E as demonstrated by flow cytometry. Two colour dot plots afterstaining CD33⁻ myeloid leukemic cells with anti-CD33-FITC andanti-CD13-PE monoclonal antibodies before and after treatment withfraction E (100.0 μg/ml) for 48 hours are shown. Isotype matched controlmonoclonal antibodies (FITC and PE labelled) were used as negativecontrols. Upper left quadrants represented CD13⁺, CD33 cells and upperright quadrants represented CD13⁺, CD33⁺ cells.

[0038]FIG. 6 represents flow diagram for example 2

[0039]FIG. 7 represents flow diagram for example 3

EXAMPLE 1

[0040] Collection of Plant Material

[0041] The leaves and all other plant parts of Piper betel werecollected from the climber from different areas and West Bengal, India.A voucher specimen was deposited at the Deptt. of Medicinal Chemistry atthe Indian Inst. of Chemical Biology, 4 Raja S. C. Mullick Road,Kolkata-700 032.

EXAMPLE 2

[0042] Fresh leaves 5.3 kgs of Piper betel is thoroughly washed withdistilled water. Leaves are cut into small pieces. It is thenhomogenized with distilled water 1.5 liter in a mixture-blender. Theextract was filtered through a fine cheese-cloth and the filtrate wascollected. The plant material inside the cloth was again extracted withwater (1000 ml). The extract was filtered in a similar way. This processof extraction was repeated three times. The combined extract wascentrifuged to obtain a clear solution. The clear extract waslyophilized yielding a semi-solid mass 150 gms. The extract was thentested for biological activity.

[0043] A potion (10.02 gm) of the extract was fractionated on SephadexLH-20 column chromatography. The column was eluted with water,water-methanol (1:1) and methanol as eluent to give three fractions,fractions 1-3, in the order of elution. The three fractions were thentested for biological activity.

[0044] The fraction-2 (designated as Fraction E) showed biologicalactivity, i.e., destruction of myeloid cells in human patients' ex vivoblood. Thin layer chromatography of this fraction showed two spotshaving Rf. 0.50 and 0.41 in the solvent system n-butanol:aceticacid:water (13:3:5). The HPLC analysis of this fraction showed severalpeaks as in FIG. 1 using Intersil ODS-3 (4.6×250 mm) analytical column,with a solvent system methanol:water:acetic acid (23:76:1), having flowrate of 1.0 ml/min and detection at 280nm. With the retention time 3.60,8.60, 9.30, 11.50, 12.70, 16.40, 19.20, 20.00, 24.00 and 36.60 minsrespectively.

[0045] Each peak of this fraction E was separated in a preparative HPLCusing μ-Bonda pak column (19×300 mm), with a flow rate 12 ml/min and thedetection at 280 nm. Compounds isolated from individual peak were testedfor biological activity as described above. A purified compound wasisolated from the peak having retention time 24.00 min. The structure ofthe compound was established as 3-O-p-coumaryl quinic acid (FIG. 2), mp.245 to 247° C., [α]_(D)−75° (methanol). IR γ^(KBr) _(max) cm⁻¹ :3379(OH), 1691 (GO), 1514, 1383, 1269, 1173 and 830 ¹H-NMR (CD₃OD) 7.64(1H,DJ = 15.9, H-7), 7.46 (2H,d, J = 8.4, H-2 and 6), 6.81 (2H,d, J =8.4, H-3 and 5), 6.35 (1H,d, J = 15.9, H-8), 5.38 (1H, m, H-3), 4.19(1H, br., H-4) 3.84 (1H, m, H-5), 2.2 (2H, br., H-2) and 2.0 (2H, br.,H-6) ¹³C-NMR (CD₃OD) 176.00 (COOH), 167.65 (C-9), 160.24 (C-4), 145.68(C-7), 130.17 (C-2 and 6), 126.25 (C-1), 115.81 C-3 and 5), 75.16(C-1'), 72.48, 70.98, 70.57 ,37.79 and 37.22 FAB MS m/z 329 (M⁺+ H), 361(M⁺+ Na), 377 (M⁺+ K)

EXAMPLE 3

[0046] The fresh leaves and all other plant parts of Piper betel (1.2kg) was homogenized with methanol (800 ml) in a mixture-blender and thensonicated in an ultrasonic bath with 3 burst each for 15 min and allowedto be extracted over night for 16 hrs. Filtering through Whatman No. 1filter paper separated the methanol-extracted material. The process ofextraction was repeated for three times. The combined extract wasevaporated to dryness in a flash evaporator under reduced pressure at40° C. The residual substance was then dried under high vacuum and thesemi-solid mass (35.22 gm) was tested for biological activity (asmentioned above).

[0047] The methanol extract (14 gm) was chromatographed on Diaion HP-20(380 gm), elution being carried out with water, water-methanol (1:1) andmethanol. Eluted three fractions were tested for biological activity.

[0048] HPTLC of water-methanol fraction (fraction E) showed similar Rfvalues (0.5 and 0.41) as described in example. Active compound,3-O-p-coumaryl quinic acid was isolated from the fraction E by usingpreparative HPLC as described in example-2.

EXAMPLE 4

[0049] Preparation of peripheral blood mononuclear cells (PBMC) fromCD33+ myeloid leukemia patients ex vivo blood.

[0050] Whole blood (10 ml) was drawn from a previously diagnosed CD33+myeloid leukemic patient and mononuclear cells were separated byFicoll/hypaqal density gradient centrifugation.

EXAMPLE 5

[0051] PBMC from CD33⁻ myeloid leukemia patients. Whole blood (10 ml)was drawn from a previously diagnosed CD33⁻ myeloid leukemia patient.Mononuclear cells were separated by Ficoll/hypaque density gradientcentrifugation.

EXAMPLE 6

[0052] Incubation of PBMC of myeloid leukemia patients with crude Piperbetel extract. PBMC (2×10⁶/ml) of myeloid leukemia patients wereincubated with Piper betel extract (10.0 mg/ml) for 48 hours and thenwashed and counted for viability.

EXAMPLE 7

[0053] Incubation of PBMC of myeloid leukemia patients with fraction Ein vitro. PBMC (2×10⁶/ml) of myeloid leukemia patients were incubatedwith varied concentrations of fraction E for 48 hours. Cells were thenwashed and counted for viability.

EXAMPLE 8

[0054] Incubation of PBMC of myeloid leukemia patients with purifiedcompound 3-O-p-coumaryl quinic acid in vitro. PBMC (2×10⁶/ml) of myeloidleukemia patients were incubated with varied concentrations of purifiedcompound for 48 h. Cells were then washed, and counted for viability.

EXAMPLE 9

[0055] Flow cytometry of myeloid leukemia cells. Myeloid leukemia cellsbefore and after in vitro incubation with crude Piper betel extract,purified fraction E or purified 3-O-p-coumaryl quinic acid were markedwith anti-CD33-FITC and anti-CD13-PE monoclonal antibodies. Cells werethen analysed in a flow cytometer (FACS Calibur, Bection Dickinson, USA)

EXAMPLE 10

[0056] Incubation of myeloid leukemic cells with fractions havingretention time 3.60 (fraction-1) and 24.00 min (fraction 9). PBMC ofmyeloid leukemic patients were incubated with above-mentioned fractions,alone or in combination of the two for 48 hrs. Cells were then washedand counted for viability.

[0057] Results of the Experiments

[0058] Results of Example 6:

[0059] As shown in FIG. 3, CD33+ myeloid leukemia cells were killed inthe presence of crude Piper betel extract. On the other hand, crudePiper betel extract has no effect on the CD33⁻ myeloid leukemic cells.

[0060] Results of Examples 7, 8 and 9:

[0061] As shown in FIG. 4, CD33+ myeloid leukemia cells are destroyed byfraction E. On the other hand, CD33⁻ myeloid leukemia cells remainedunaffected. (FIG. 5). PBMC of CD33+ myeloid leukemia patients (both AMLand CML) are destroyed by purified compound 3-O-p-coumaryl quinic acid(Table -1).

[0062] Results of Example 10:

[0063] As shown in Table 2 combining fractions of 1 and 9 producedgreater effects as compared to their individual activity at lowerconcentration. TABLE 1 Growth inhibition of AML and CML cells in vitroby purified compound Percent inhibition of growth by purified compound3-O- p-coumaryl quinic acid Type of myeloid leukemia 100 μg/ml 600 μg/mlAML 53.37 100.00 CML 2.53 79.50

[0064] TABLE 2 Growth inhibition of AML cells in vitro by fractions 1and 9 Percent inhibition of growth Doses Type of myeloid leukemiaFractions (μg/ml) % Inhibition AML 1 100 79.50 9 100 54.00 1 + 9 50 + 50100.00

We claim:
 1. A method of treating either CD33+ acute myeloid leukemiaCD33+ chronic myeloid leukemia in mammals including humans, using atleast one of water:methanol fraction nos. 1 and 9, which have beenobtained from a polar extract of the plant piper betel by HPLC, with aretention time of 3.6 and 24.0 minutes rescpectively, wherein at leastone fraction is used either individually, or in combination, said methodcomprising the steps of administering an effective amount of at leastone fraction intravenously to a patient suffering from myeloid leukemia.2. A method as claimed in claim 1, wherein a ratio of water to methanolranges between approximately 1:5 to 5:1.
 3. A method as claimed in claim1, wherein fraction no. 9 is used which essentially comprises thecompound 3-O-p-coumaryl quinic acid.
 4. A method as claimed in claim 3,wherein the compound 3-O-p-coumaryl quinic acid of fraction no. 9 isused in an effective amount in acute myeloid leukemia.
 5. A method asclaimed in claim 3, wherein the compound 3-O-p-coumaryl quinic acid offraction 9 is purified, and an effective amount exhibits about 100%efficacy against acute myeloid leukemia, at a concentration of about 600μg/ml.
 6. A method as claimed in claim 1, wherein a dosage of aneffective amount of at least one fraction individually ranges between200-500 mg/day.
 7. A method as claimed in claim 1, wherein a dosage ofan effective amount of fraction nos. 1, and 9 in combination rangesbetween 125 to 370 mg/day.
 8. A method as claimed in claim 1, whereinthe ratio for a dosage of an effective amount of fraction nos. 1 and 9ranges between 0.1:5 to 5:0.1, and is preferably about 1:1.
 9. A methodas claimed in claim 1, wherein an effective amount of fraction 1 is moreefficient as compared to an effective amount of fraction
 9. 10. A methodas claimed in claim 1, wherein an effective amount of a combination offractions 1 and 9 is more efficient in the management of myeloidleukemia as compared to an effective amount of each fraction alone. 11.A composition useful for treating either CD33+ acute myeloid leukemia orCD33+ chronic myeloid leukemia in mammals including humans, saidcomposition comprising water:methanol fraction nos. 1 and 9, which havebeen obtained from a polar extract of the plant piper betel by HPLC,with a retention time of 3.6 and 24.0 minutes respectively, wherein atleast one fraction is present either individually or in combination. 12.A composition as claimed in claim 11, wherein the fraction 9 essentiallycomprises the compound 3-O-p-coumaryl quinic acid.
 13. A method ofisolating biologically active fraction nos. 1 and 9 from thewater:methanol fraction, which have been obtained from a polar extractof the plant piper betel, said method comprising the steps of: a.cutting the plant parts into small pieces, b. homogenizing the saidpieces with polar solvent to obtain an extract, c. filtering the extractto collect filterate, d. lyophilizing the clear extract solution toobtain a semi-solid mass, e. fractionating the said semi-solid massusing column chromatography with only water, water-methanol with ratioranging between 1:5 to 5:1, and only methanol, as eluents, f. selectingwater:methanol fraction from the column chromatography, g. running HPLCwith flow rate of 1.0 ml/min, using solvent system ofmethanol:water:acetic acid of ratio about 23:76:1, h. detecting about 12peaks at about 280 nm, with varied retention time ranging between 3.6 to36 minutes, i. separating the said peaks in a preparative HPLC with flowrate of 12 ml/min, and j. obtaining fraction nos. 1 and 9 having desiredbiological activity.
 14. A method as claimed in claim 13, wherein thefraction 9 essentially comprises compound 3-O-p-coumaryl quinic acid.15. A method as claimed in claim 13, wherein the filtrate is centrifugedto obtain a clear solution before the fractionating step.
 16. A methodas claimed in claim 13, wherein ratio of dosage of fraction nos. 1 and 9ranges between approximately 0.1:5 to 5:0.1, and preferably is 1:1. 17.A method of treating either CD33+ acute myeloid leukemia or CD33+chronic myeloid leukemia in mammals including humans, using compound3-O-p-coumaryl quinic acid obtained from the fraction 9 of polar extractof piper betel by HPLC, with retention time of about 24.0 minutes, saidmethod comprising steps of administering an effective amount of thefraction intravenously to a patient suffering from myeloid leukemia. 18.A method as claimed in claim 17, wherein an effective amount of thecompound 3-O-p-coumaryl quinic acid exhibits about 100% efficacy againstacute myeloid leukemia, at a concentration of about 600 μg/ml.
 19. Amethod as claimed in claim 17, wherein an effective amount of thecompound 3-O-p-coumaryl quinic acid exhibits about 85% efficacy againstchronic myeloid leukemia, at a concentration of about 600 μg/ml.
 20. Amethod as claimed in claimed in claim 17, wherein an effective amount ofthe compound 3-O-p-coumaryl quinic acid exhibits about 60% efficacyagainst acute myeloid leukemia, at a concentration of about 100 μg/ml.21. A method as claimed in claim 17, wherein an effective amount of aconcentration of compound 3-O-p-coumaryl quinic acid ranges between50-700 μg/ml.